Our interdisciplinary research team is uniquely suited to study microRNA (miRNA) in adult stem cells. The PI'laboratory studies skin adult stem cells and their roles in skin homeostasis and melanocyte development;whereas the co-PI is leading a computational biology lab with focus on the functional analysis of miRNAs. The PI and co-PI have yet collaborated or published together. The proposed studies will utilize the strength of both PIs and the synergy will facilitate the miRNA research in adult stem cells and melanocyte development. Despite emerging evidences that miRNAs play an important role in stem cell self- renewal and differentiation, their role in stem cells has just begun to be understood and their function in neural crest stem cells (NCSCs) and melanocyte development has yet to be explored. In this revision proposal, we plan to explore the expression of miRNA during differentiation of NCSCs to melanocytes by deep sequencing method and then study whether modulation of selected miRNA expression may increase the efficiency of NCSC differentiation to melanocytes. miRNA studies were not proposed in the parent grant. The new proposal will add a new dimension to the parent grant by addressing the functions of miRNAs during NCSC differentiation which may allow us to discover potential novel mechanisms underlying adult stem cell self-renewal and differentiation. In addition, we will not limit our research using established microarray platforms to study known miRNA and mRNA, but explore the expression of miRNA and miRNA binding sites in the 3'untranslated region (UTR) in adult stem cells and melanocytes using deep sequencing methods. The ultra high-throughput sequencing method will allow us to quantify the absolute copy number of specific known miRNAs and to discover novel miRNAs. It has been suggested recently that 3'UTR length may influence the biological function of miRNAs, and we will also interrogate the interaction between miRNA and UTR during stem cell differentiation. Combining deep sequencing of both miRNA and total RNA will gain insight on how miRNAs function in stem cell self-renewal and differentiation. We expect our research will generate essential information on the function of miRNA in adult stem cells self-renewal and differentiation as well as melanocyte development. PUBLIC HEALTH RELEVANCE: These proposed studies will broad our fundamental knowledge about the function of microRNA in human neural crest stem cells (hNCSCs) and pigmented cell development, and increase our ability to selectively produce differentiated cell types from hNCSCs for regenerative medicine.